Spermatogenesis is a dynamic process of cellular differentiation that is marked by dramatic changes in patterns of gene expression. For many years we have studied the spermatogenesis-specific Pgk2 gene as a model of a tightly regulated tissue-specific gene expressed exclusively in meiotic and postmeiotic spermatogenic cells. We have exploited the uniquely accessible spermatogenic cell lineage to obtain purified populations of specific spermatogenic cell types at premeiotic, meiotic, and postmeiotic stages. This has allowed us to examine molecular parameters associated with mechanisms of transcriptional regulation, and to determine a developmental order of molecular events that lead up to initiation of transcription of the Pgk2 gene. This has allowed us to formulate testable hypotheses about the mechanisms involved in this regulatory process. In this application, we propose experiments to identify key factors involved in the regulation of this gene, to characterize changes in chromatin composition and structure that precede transcriptional activation of this gene, and, finally, to assess the mechanism by which a tissue- and gene-specific demethylation domain develops in this gene prior to any other changes in transcriptional parameters and to assess the extent to which demethylation is required to predispose transcriptional activation of the Pgk2 gene in vivo. These experiments will allow us to define cause-and-effect relationships among regulatory events associated with transcriptional activation of a spermatogenesis-specific gene. This will lead to an enhanced understanding of the mechanisms that regulate tissue-specific gene expression in general, and spermatogenesis-specific gene expression in particular.